Composition and method for treatment of vaginal yeast infections

ABSTRACT

A composition for the treatment of vaginal yeast which promotes normal vaginal bacterial growth during treatment, comprising a yeast-inhibitory agent selected from one or more of the group consisting of ethylene glycol, propylene glycol, and glycerol, an effectively buffered aqueous acetate solution and cells of a Lactobacillus species. In the composition the concentration of acetate is effective to establish a pH of the composition in the range of from about 5 to about 7, the presence of unionized acetate inhibits yeast selectively compared to the bacteria, and the proportions of the yeast inhibitory agent and the buffering solution are effective to allow the active yeast-inhibitor agent to inhibit the vaginal yeast Candida. A method of treatment of vaginal yeast while promoting normal vaginal bacterial growth, by intravaginal application of the composition described above, is disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of Ser. No. 08/892,615 filedJun. 1, 1992, now U.S. Pat. No. 5,340,836, which is a continuation ofSer. No. 07/657,522 filed Feb. 19, 1991 (now abandoned).

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to compositions having utility for thetreatment of vaginal yeast while promoting normal bacterial growth, andto a method of treatment of vaginal yeast conditions.

2. Description of the Related Art

Yeast infections, also termed Candidiasis, moniliasis, or Moniliavaginitis, are among the most frequently occurring of vaginalinfections. These vaginal infections are caused by fungus-like yeasts ofthe genus Candida (formerly Monilia), and specifically the speciesincluding C. albicans, C. tropicalis, C. psuedotropicalis, and C.stellatoidea. These Candida fungal microorganisms, also known asMonilia, normally grow and thrive at low concentrations in the mouth,gastrointestinal tract, and vagina, coexisting with the normalbiological flora in these regions, without adverse effect on health orphysiological function.

In the vaginal the homeostasis between these Candida fungi and thenormal vaginal flora nonetheless may be upset by changes in vaginal pH,or other conditions upsetting the vaginal equilibrium or otherwiselowering resistance to infection. Monilia overgrowth then may occur,resulting in a thick, white colored vaginal discharge which often islikened to cottage cheese in consistency, and associated with a strongodor. This condition occurs frequently in women during pregnancy, or asa result of conditions such as diabetes, treatment with antibiotics,steroids or immunosuppressive therapy, or hormonal changes occurringduring the menstrual cycle.

The two major classes of antifungal agents employed are the polyenes(nystatin and amphotericin) and the imidazole derivatives (miconazole,clotrimazole, butuconazole, and ketoconazole).

Over the counter preparations for vaginal cleaning and deodorizationgenerally contain glacial acetic acid and water and may contain glycerinand various thickening agents. Although vinegar douches comprising asolution of vinegar and water have been commonly employed for many yearsfor cosmetic douching purposes, and are often resorted to for flushingyeast deposits and bacteria from the Monilia-infected vagina, suchmethods do not in fact destroy the Candida fungi in situ. The pH ofthese products are all at or below 3.5 when measured on a pH meter. Thenormal vaginal pH is approximately 4.5 and this homeostatic condition isnot significantly altered by vinegar douching. vinegar douchingtreatment has been predicated on the incorrect belief that lower pHvalues in the vagina promote the growth of normal vaginal flora at theexpense of Candidal fungal species. In the vaginal pH range, thepathogenic yeasts compete with normal bacterial flora, displacing orsuppressing the growth of biota needed for healthy vaginal functioning.

More effective treatment of Candida infections typically involves theapplication of an anti-yeast medication to the vagina, with intravaginaladministration of medicine being repeated at selected intervals over aperiod of days or weeks.

An example of an intravaginally applied medication is a water misciblecream composition containing 2% by weight1-2,4-dichloro-beta-(2,4-dichlorobenzyloxy) phenethyl] imidazole nitratein a cream base comprising mineral oil, pegoxol-7 stearate,peglicol-5-oleate, butylated hydroxyanisole, benzoic acid, and water,commercially available from Ortho Pharmaceutical Corporation, Raritan,N.J., under the trademark MONISTAT 7. This composition is recommended bythe manufacturer to be applied intravaginally once daily for 7 days.

It is an object of the present invention to provide a Candidastaticand/or Candidacidal composition which promotes a high rate of growth ofnormal vaginal biota during treatment, while selectively inhibiting thegrowth, reproduction and germination of Candida organisms in the vagina.

Other compositions used to treat Candia, and other uses for thecomponents or related components of Applicant's composition arediscussed below.

U.S. Defensive Publication T937,002 of N. G. Baker discloses thetreatment of ungulate mastiris, a bacterial infection, with glycolshaving 3 to 4 carbon atoms and at least one terminal group, e.g.,1,3-butylene glycol, administered in pharmacologically effective doses.The disclosure of this reference and of all others cited herein isincorporated herein by reference.

U.S. Pat. No. 4,310,510 to K. N. Sherman, et al. discloses anintravaginally applied anti-fertility composition comprising a foamableaqueous emulsion containing progesterone as the active ingredient. TheSherman, et al. patent at column 8, lines 48-68 discloses that theformulation, in addition to progesterone and an anionic surfactant, maycontain buffering agents such as phosphates, citrates or tartrates, tomaintain a pH in the range normal in the female genital tract, that ison the order of about 4.5 in the vagina. In a preferred disclosedembodiment, the composition contains 10-50% by weight of a lower glycolas a foam stabilizer, the patent stating that in such concentrationrange, the glycols prevent microbial growth in the formulation so thatpreservatives are not required. In addition, other foam stabilizers maybe substituted for glycols, such as glycerol and the polyalkyleneglycols, such as polypropylene glycol and polyglycerols.

Among the progesterone compounds useful in the practice of Sherman'santifertility composition (column 6, lines 11-30 of Sherman) are variousprogestin acetate compounds. There is no indication in Sherman of anyCandidacidal activity of glycols and/or a non-steroid acetate compoundnor of their use in the absence of progesterone and anionic surfactants.

U.S. Pat. No. 3,970,759 to J. W. Frankenfeld, et al. discloses theinhibition of bacteria, yeasts, and molds in cosmetic personal carecompositions containing edible oils and organic nitrogen compounds, byincorporating therein an effective amount of a linear aliphatic1,3-diol, or a monoester or diester thereof. The diols disclosed assuitable for this purpose contain 5-15 carbon atoms in the chain;suitable diesters have a linear aliphatic acyl group of 2-20 carbons.

U.S. Pat. No. 4,294,852 to R. H. Wildnauer, et al. discloses a topicallyapplied skin composition for treatment and control of dry skindisorders, comprising an aqueous phase in which is dissolved one or morespecified aliphatic alcohols in combination with one or more selectedaliphatic mono, di- or tri- carboxylic acids having 2-10 carbon atoms,including those which are substituted at either or both of the alpha andbeta carbons with a hydroxyl or keto functional group.

The Wildnauer, et al. patent discloses at column 5, lines 6-7 that thedisclosed composition may comprise propylene glycol as an additive.Propylene glycol is also mentioned in the same column at lines 17 etseq. as an active compound with which the disclosed composition may beemployed as a vehicle. Other such active ingredients includedexamethasone acetate and anti-fungal agents such as griseofulvin,Mycostatin™, miconazole and minconazole nitrate. Among the alpha,beta-dihydroxy carboxylic acids disclosed in the patent are glycericacid; the disclosed keto acids include glyoxylic acid, pyruvic acid andacetoacetic acid.

U.S. Pat. No. 3,836,672 to D. D. Wright, et al. discloses the treatmentof bacterial and fungal infections by linear diols and/or theirmono-esters, the diols containing 7-15 carbons and hydroxy groups on thefirst and third carbon atoms while the mono-esters have acyl groups of2-10 carbons.

The Wright, et al. patent discloses in the paragraph bridging columns 4and 5 that Monilial vaginitis caused by Candida albicans is treated tocontrol the fungal organism with minimum disturbance of the normalvaginal bacterial flora. A 50-50 mixture of 1,3-nonanediol and1,3-butanediol monooctanoate is disclosed as effective for the treatmentof fungal vaginitis, and as being effective against fungi at levelswhere it is only mildly inhibitory to bacteria. The patent at column 6,lines 30-38 describes inert carriers for the disclosed compositions,including glycerol as an inert carrier for providing a paste orcream-like consistency, or propylene glycol or ethylene glycol as aninert carrier for propellant type or aerosol materials.

U.S. Pat. No. 3,821,413 to H. L. Hellyer, Jr., discloses atmosphericgermicidal compositions comprising from about 5-85% by weight of aglycol germicide, e.g., ethylene glycol, or propylene glycol, from 2-40%by weight of an organic polar coupling compound including variousspecified alcohols such as beta-phenylethyl alcohol and alphaterpineol,and from about 5-80% by weight of an organic, relatively non-polarcompound for forming hydrophobic micelles surrounding the glycolmolecules in the mixture to reduce the glycol affinity to atmosphericmoisture. The non-polar compounds disclosed in the patent at column 2,lines 54-58 include various acetate compounds such as benzyl acetate,citronellyl acetate, octyl acetate, and 1,3-nonanediol acetate.

U.S. Pat. No. 3,384,541 to W. G. Clark discloses a spermicidal vaginalfoam-producing composition comprising ethoxylated tallow alcohol,propylene glycol or other glycols or glycerol. The patent discloses toutilize such composition in combination with a buffered fructosesolution. Example 23 of the patent teaches to add iodine compounds tothe composition for the treatment of vaginitis, and Candida albicans,Monilia, and Trichomonas vaginalis, along with the contraceptivefunction of the composition.

U.S. Pat. No. 2,451,955 to H. Keil, et al. discloses the use of acomposition to determine susceptibility to dermatitis, comprising asolution of a synthetic vicinal alkyl pyrocatechol with 10-18 carbons inthe alkyl group, in a non-irritant organic solvent such as iso-amylacetate, a water insoluble acetate ester.

U.S. Pat. No. 2,467,884 to N. N. Elias discloses spermicidalcompositions comprising neutral aliphatic hydroxy compounds such ashigher fatty alcohols, hydroxyethers, hydroxyesters and hydroxyesterethers. The composition may comprise an aqueous vehicle containing awater soluble acid for bringing the pH of the resulting compositionwithin the range of 1-7. The patent discloses that the pH is establishedby means of whatever simple organic or inorganic acids and buffer saltsmay seem desirable. The patent discloses that glycerine may be added tothe spermicidal compositions to prevent their dehydration and freezing.The patent also discloses the use of water soluble acids such as aceticacid, with potassium acetate and sodium acetate being disclosed asbuffers.

When Saccharomyces yeast cells are incubated in hypertonic glycerolsolutions, the cells shrink to about 40% of their original volume withglycerol premeating the cells resulting in alteration of membranemorphology. The shrinking is reversible upon removal from the 8.7%glycerol solution but incubation in solutions above 17.4% glycerol islethal for exponentially growing yeasts. Protoplasma 92:177 (1977).

Glycerol is also known to be metabolized by yeasts as Candida andSaccharomyces and is a by-product of some yeast metabolism. See EuropeanJ. Blochem 5: 165 (1968).

SUMMARY OF THE INVENTION

In a broad aspect, the invention relates to a composition having utilityfor the treatment of vaginal yeast while promoting normal vaginalbacterial growth, comprising a solution comprising one or more medicallyacceptable agents, said agents selected from the group consisting ofethylene glycol, propylene glycol, glycerol, acetate derivatives ofglycerol, and acetate; said solution having active yeast inhibitoryactivity and being effectively buffered to establish the pH of thecomposition at about neutral.

In a narrower aspect, the present invention relates to a compositionhaving utility for the treatment of vaginal yeast while not adverselyaffecting normal vaginal bacterial growth, comprising:

(a) a medically acceptable active yeast-inhibitory agent selected fromone or more members of the group consisting of ethylene glycol,propylene glycol, and glycerol, having an effective concentration at thevaginal treatment site of at least 8% by volume of the compositionvolume plus the volume of other liquids present at the treatment site;and

(b) an effectively buffered aqueous acetate solution constituting 92% orless by volume of the composition;

wherein the concentration of acetate in said effectively bufferedaqueous solution is effective to establish the pH of the composition inthe range from about 5.0 to about 7, and

wherein the active yeast-inhibitory agent and said effectively bufferedaqueous acetate solution are in selected proportions relative to oneanother effective to allow the active yeast-inhibitory agent to beinhibitory against vaginal yeast of the genus Candida.

The composition may further comprise an antibiotic such as Nystatin,imidazoles or others, in addition to the medically acceptable agents,for example, in addition to the active yeast-inhibitory agent and theacetate solution.

In another aspect the invention relates to a method for treatment ofvaginal yeast while not adversely affecting normal vaginal bacterialgrowth, comprising applying to the vaginal locus a buffered solution ofa yeast-inhibitory agent in an amount effective to inhibit Candida, saidyeast-inhibitory agent selected from one or more members of the groupconsisting of ethylene glycol, propylene glycol, and glycerol, saidsolution buffered to maintain the vaginal pH in the range of from about5 to about 7, for sufficient time to permit the yeast-inhibitory agentto inhibit the vaginal yeast while permitting normal bacterial growth tooccur in the vaginal locus, and permitting the vaginal bacteria toconsume vaginal nutrients and competitively inhibit growth of yeasts.

The invention also comprises a method of allowing normal vaginal florato be restored, comprising douching with a buffered solution of ayeast-inhibitory agent, said yeast-inhibitory agent selected from one ormore members of the group consisting of ethylene glycol, propyleneglycol, and glycerol, said solution buffered to maintain the vaginal pHin the range of from about 5 to about 7, for sufficient time to permitflushing of the vagina while promoting normal bacterial growth in thevagina; and a method of allowing normal vaginal flora to be restored,comprising douching with an effectively buffered aqueous acetatesolution, said solution buffered to maintain the vaginal pH in the rangeof from about 5 to about 7, for sufficient time to permit flushing ofthe vagina while encouraging normal bacterial growth in the vagina.Applying to the vaginal locus an amount of the above-describedcomposition effective to inhibit Candida.

The inhibition of Candida comprises a static effect at lowerconcentrations of glycol or glycerol and a cidal effect atconcentrations near or above 40%. The term "inhibit", "inhibitory" or"inhibition" herein means decreasing the rate of growth and shall alsoinclude any killing of yeast cells that may occur at higherconcentrations and/or less desirable growth conditions for the Candida.

Other aspects and features of the invention will be more fully apparentfrom the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of optical density (A₆₀₀) of Lactobacillus versus mMconcentration of propylene glycol (PG), urea (U), ethylene glycol (EG),and NaCl (NaCl).

FIG. 2 is a graph of percent suppression of Candida at variousconcentrations of diacetin (D), methylacetate (MA), ethylacetate (EA)and triacetin (T).

FIG. 3 is a graph of optical density (A) of Candida versus per centadded serum at 0%, 10%, and 20% glycerol.

FIG. 4 is a graph of optical density (A₆₀₀) of Streptococcus (), E.coli (x) and Lactobacillus (Δ) at different pH values of acetate after23-30 hours.

FIG. 5 is a graph of optical density (A₆₀₀) of C. tropicalis atdifferent pH values of acetate at 24 (Δ) and 48 () hours.

FIG. 6 is a graph of optical density (A₆₀₀) of C. stellatoidea atdifferent pH values of acetate at 27 (open triangles) and 48 (closedcircles) hours.

FIG. 7 is a graph of optical density (A₆₀₀) of C. albicans (strain14053) at different pH values of acetate at 31 (open triangles) and 46(closed circles) hours.

FIG. 8 is a graph of optical density (A₆₀₀) of S. cerevisiae atdifferent pH values of acetate at 24 (open triangles) and 43 (closedcircles) hours.

FIG. 9 is a graph of the relative number of microorganisms in thesuccession of Candida and Lactobacillus showing Candida (control-closedcircles; acetate only-closed upward pointing triangles; acetate plusglycerol-closed downward pointing triangles), and Lactobacillus(corresponding open figures).

DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

The present invention is based on the fact that the normal pH balance ofthe vagina, being typically slightly acidic on the order of pH 4.5, is ahomeostatic condition with respect to the populational dynamics ofnormal vaginal biota. As indicated hereinabove in the "Background of theInvention" section, the prior work in the treatment of Candidainfections has typically sought to effect vaginal treatment at thenormal vaginal pH acidic conditions, on the theory that compositionswhich were inhibitory or cidally effective against Candida at suchnormal pH conditions would permit the normal vagina biota to bereestablished concurrent with the destruction of the fungal infection.

The present invention is based on the discovery that growth of vaginalbiota concurrent with destruction of Candida organisms is best effectedat a neutral pH, inasmuch as the rate of growth of normal vaginal biotais substantially higher at such elevated pH conditions than at the lowerpH values characteristic of the healthy vagina at equilibrium. Thecomposition of the invention is based on the surprising finding thatwith the composition of the invention, a succession of environmentalchanges in the vagina provides maximum yeast inhibition while enhancinggrowth of the normal vaginal flora. Thus, laboratory work shows that theglycol compounds such as glycerol, propylene glycol and ethylene glycol,which are nontoxic to human cells and bacteria, inhibit the yeast at theinitial roughly neutral pH of the composition (e.g. above pH 4.5,preferably about pH 5 to pH 7, and most preferably at about pH 7.0). Asbacterial growth occurs, vaginal nutrients are consumed, and the pHdecreases, the acetate component of the composition inhibits the yeastto a greater extent than it inhibits the normal vaginal bacterialpopulation.

Accordingly, the compositions of the invention effect destruction of theyeast organisms by being biostatically or biocidally effective againstCandida at a neutral pH secured by an appropriately constituted aqueousacetate buffer solution. Thus, the topical application of compositionsof the invention to the vaginal locus will expose the vaginal tissue toa neutral pH where growth of normal vaginal biota is promoted,concurrent with the inhibition of the undesired Candida organisms.

Concurrent with this accelerated growth of the desired normal vaginalbiota at neutral pH, the Candida and other yeast species are similarlymetabolically accelerated, to the extent that such organisms are notkilled by the active yeast-inhibitory component of the composition ofthe invention. While it may be initially thought that such accelerationof the metabolic growth rate of the yeast species would be deleteriousin the sense that an increased population would require cidal action bythe composition, the results support the conclusion that the metabolicbyproducts of such yeast organisms include metabolites such as glycerol,and glycols, e.g., ethylene glycol and propylene glycol, whichthemselves are biostatically or biocidally effective against the Candidaand potentially other yeast species. Thus, the undesired yeast growthmay be effectively poisoned by its own metabolic end products, incombination with similar external end products of the normal vaginalmicroorganisms. In fact, in the population growth of such yeast speciesin culture, the population in a nutrient-rich solution of constantconcentration will experience log growth to a point of metabolichomeostasis, where the available nutrients and deleterious metabolitescome into equilibrium with one another, followed by declining microbialconcentration as the metabolites accumulate and reduce the population.

Thus, results indicate that the accelerated production of metabolicbyproducts as effective species in inhibiting Candida generated in situin the vaginal environment, in combination with thefungistatic/fungicidal action of the composition of the invention,produces a highly effective treatment of the Candida organisms and otheryeast species, resulting in their control and/or destruction.

With this bifurcated action against the vaginal yeast, the pH conditionsmaintained by the acetate buffer promote the growth of normal vaginalflora. As such normal flora proliferates, the metabolic waste productsof the normal vaginal flora, which may include the aforementionedmetabolic byproducts effective against the Candida and other yeastspecies, gradually accumulate to overcome the buffering action of thecomposition, and return vaginal acidity to normal pH conditions.

As the buffering action is overwhelmed, and lower pH, in the rangeapproaching normal acidity of about 4.5, is established, the acetateconstituent of the buffering solution is increasingly nonionized (itsionization decreasing with decreasing pH), and the resulting increasedconcentration of acetic acid and other nonionizable esters of acetate,such pH equilibrium effects increase the inhibitory efficiency of thecomposition against the yeast. The extent of this ancillary inhibitoryaction of the composition at reduced pH values in the vicinity of normalvaginal pH is dependent on the specific acetate material utilized in thecomposition.

Generally, any suitable acetate compound may be employed as the acetateconstituent of the buffer solution, including compounds of the formulaCH₃ COOR, wherein R is hydrogen or a lower alkyl. Among the variousacetate compounds of such type, acetic acid (CH₃ COOH) is preferred,followed by C₁ -C₃ alkyl acetate compounds. Although any physiologicalinert buffer in the neutral pH range would promote normal vaginal floragrowth, the present invention is directed in a preferred aspect to theuse of acetate buffers, for the reason discussed above that they have anincreased biological activity against Candida and other yeasts asvaginal pH is reduced to normal levels on the order of about 4.5.

Acetate compounds with which the acetate moiety is locked into anonionized form may be used. For example, triacetin and diacetin(glycerol compounds esterified with acetic acid) are capable of servingboth as the active yeast-destructive agent as well as the acetatesource.

The active yeast-inhibitory agent, preferably as selected from one ormore of ethylene glycol, propylene glycol, and glycerol, may in thebroad practice of the invention preferably constitute from about 8% toabout 40% by volume of the composition as delivered to the treated site,based on the total volume of the active yeast-inhibitory agent and theeffectively buffered solution and any dilution of liquids in theadministration of the agent. Higher concentrations are effective but mayresult in tissue irritation. The preferred concentrations provideeffective Candida inhibition while not being irritating although higherconcentrations also provide effective inhibition of Candida.

Correspondingly, the effectively buffered aqueous acetate solution mayconstitute from about 60% to about 92% by volume, on the same totalvolume basis. The concentration of acetate compound in the effectivelybuffered solution should be sufficient to establish the pH of thecomposition above 4.5, that is, above the normal vaginal pH. Preferablythe pH of the composition is in the range of from about 5 to about 7,and the active yeast-inhibitory agent and buffering solution should eachbe in selected proportions relative to one another which are effectiveto allow the active yeast-inhibitory agent to be biocidally effectiveagainst Candida organisms. Most preferably, the pH is about neutral. Aconcentration of about 100 mM has been found to be effective.

It will be appreciated that more than one of the aforementioned activeyeast-inhibitory compounds may be used in combination in compositions ofthe invention, e.g., a mixture of propylene glycol and glycerol.

While the concentrations of the glycol or glycerol component and thebuffering acetate compound may be widely varied in the composition andrelative to one another; within the foregoing functional constraints(i.e., the buffering solution being effective to establish a pH in therange of from about 5 to about 7, and the active yeast-inhibitory agentbeing in sufficient concentration and proportion relative to thebuffering solution to allow it to be biostatically effective against thevaginal yeast), buffering solutions containing about 100 millimoles ofacetic acid per liter in combination with propylene glycol present at aconcentration of 1.0 to 1.5 Molar have been found to be generallyeffective Candidacidal compositions. Generally, glycerol when used asthe active yeast-inhibitory agent may be used at preferredconcentrations on the order of 1 Molar based on the total volume of theglycerol and the buffer solution. In the case of propylene or ethyleneglycol, concentrations on the order of 1.5 Molar, on the same totalvolume basis, may be advantageously employed for killing yeast but maybe unacceptably effective in also killing vaginal bacteria.

With reference to the glycolic yeast-inhibitory agents, as selected fromone or more members of the group consisting of propylene glycol,ethylene glycol, and glycerol, all are polyhydroxy-functional compounds.It is possible that other active yeast-inhibitory agents may be employedin place of these compounds, to provide advantages similar to thoseobserved with the above-described specific agents. Nonetheless, from thestandpoint of physiological compatibility, and inhibitory activity,these materials are highly preferred.

It is to be understood that the composition of the invention hasCandidacidal activity in the absence of any medically active orantibiotic compounds other than the acetate and glycol compounds, butother Candidacidal drugs may be added. Nystatin, a polyene anti-fungalantibiotic complex produced by various Streptomyces species which hasbeen used to inhibit fungi and yeasts, or other anti-fungal agents, maybe added to the composition of the invention to provide enhancedanti-Candida activity. Other fungal agents that may be added include,but are not limited to clotrimazol, miconazole, butoconazole andterconazole.

It is anticipated that in the use of the composition of the inventionfor Candidacidal treatment, the composition would not contain compoundsunrelated to or unrequired for such treatments such as hormones,contraceptives, non-glycol alcohols, other organic molecules orantibacterial compounds which are not primarily Candidacidal, norsignificantly larger glycol or acetate compounds.

The fungicidal or fungistatic compositions of the invention mayadditionally contain any suitable additives which do not preclude itsyeast-inhibitory character and the neutral pH vaginal floragrowth-promotion mechanism previously described. Illustrative ofexemplary additives are stabilizers, extenders, antioxidents, perfumes,dyes, fillers, surfactants, etc.

In order to enhance the in vivo retention of the composition after itsintravaginal application, the composition may suitably contain apharmaceutically acceptable carrier, or a thickener, providing thecomposition with sufficient viscosity to be appropriately retaineddespite movement and position of the female to whom it has beenadministered. Suitable thickeners of such type may include materialssuch as hydroxypropylcellulose and/or hydroxypropylmethylcellulose. Aparticularly preferred thickener is carboxymethylcellulose, ascommercially available under the trademark CARBOPOL® (HerculesCorporation, Wilmington, Del.).

As a class, nitrogen cross-linked carboxyl-containing thickeners arepreferred. The addition of thickeners is highly desirable to provide theaforementioned retention function for the composition after itsintravaginal administration. In the absence of such thickeners, orpharmaceutically acceptable carriers of other types, providing suchviscosity and/or retention function, the composition may have to beapplied more frequently than would be the case when a thickener orcarrier is employed.

The composition of the invention may be applied to the vaginal area byany method known in the art, for example, as a sponge, as an osmoticdevice, as a douche, and so forth for treatment of vaginal yeast and toallow the normal flora to be maintained and the yeast population andgrowth to be repressed.

The features and advantages of the invention are more fully shown withrespect to the examples set forth below. In these studies thecomposition of the invention is tested in laboratory tests in which theculture tube simulates the vagina.

In ovulating women, the age group which gets vaginitis, the vagina has avery thick, impervious layer of squamous epithelial cells, much like aglass wall of a culture tube. The vagina and cervix secrete very littlefluid under normal conditions, thus there is no flow of material. Thereis usually only 1-2 ml of cervical secretion present and it is staticmuch as the culture fluid is in a tube. The walls of the vagina are notrigid and thus collapse together in a convoluted shape that gives thevagina an enormous surface to volume ratio not present in a cylinder.But with no gas or fluid exchange due to the epithelium, this ratio isnot relevant for these studies. The O₂ tension in the vagina and theculture tube are similar and undergo similar changes when bacteriaand/or yeast are metabolizing the nutrient. Medium 19 used in nearly allof these studies has an electrolyte, protein and carbohydratecomposition very similar to normal vaginal fluid. The experiments withadded serum mimic and carry to about 4-fold excess any changes thatmight occur during any inflammatory exudation.

With respect to these examples as hereinafter set forth, the followingmethods and procedures were employed:

Isolation of Candida sp and Normal Vaginal Bacterial Flora from ClinicalSpecimens.

Specimens were collected from women clinically diagnosed as havingCandida vaginitis at the Pitt County Health Department, Greenville, N.C.The presence of yeast was confirmed by microscopic examination of wetpreparations by the staff at the Health Department. Specimens were alsocollected from asymptomatic women attending the Family Planning Clinicat the Pitt County Health Department. All specimens were submitted onCulturette® swabs and visibly contained exudates typically associatedwith Candida vaginitis. The specimens were streaked on chocolate agar,blood agar, and Sabouraud's dextrose agar, (peptone, 10 g; glucose, 40g; agar 15 g; 1000 ml water; pH 5.6), with 40 ug/ml chloramphenicol. Thespecimens remaining on the swabs were incubated in trypticase soy broth.All cultures were incubated at 37° C in 5% CO₂ atmosphere for 48 hr.Well isolated colonies were selected for subculture. After three cyclesof subculturing, smears of the isolates were Gram stained,microscopically examined, and morphologically characterized. Theisolated organisms were stored on slants at 4° C. until furthercharacterized or used experimentally.

Medium 19

In the subsequent examples, Medium 19 refers to the nutrient compositionset forth in Table I below.

                  TABLE I                                                         ______________________________________                                        Peptone                 9.4    g                                              Yeast Extract           4.7    g                                              Beef Extract            2.4    g                                              NaCl                    10.0   g                                              Dextrose                10.0   g                                              Agar                    23.5   g                                              Water qs                1000   ml                                             pH 6.1 ± 0.1 after sterilization                                           Medium 19 broth is Medium 19                                                  with the Agar deleted.                                                        ______________________________________                                    

Lactobacillus

The Lactobacillus species were grown in Lactobacilli MRS Broth purchasedfrom Difco Laboratories, Detroit, Mich.

Growth Assay for Candida

A culture tube containing 10 ml Medium 19 broth was inoculated with C.albicans and incubated overnight at 37° C. Two drops of the heavyovernight growth were diluted into a fresh tube of Medium 19 broth andincubated at 37° C. until the yeast was in active logarithmic growth(about 4 hr). This logarithmic phase culture was diluted to A₈₀₀ =0.05in Medium 19 broth for use as experimental inoculum. As used herein,A₆₀₀ refers to the culture sample absorbance (turbidity) measured at 600nanometers wavelength incident light, as passed through the sample anddetected by a suitable photodetector.

The experimental tubes were prepared to contain a total volume of 9.9 mlmedium and reagents. Then 0.10 ml of microbial inoculum was added. Theinoculated medium was vigorously mixed, and then the tubes wereincubated as stationary cultures at 37° C for the duration of theexperiment.

The amount of growth was determined turbidimetrically at appropriateintervals for 96 hours. The culture tubes were vigorously mixed touniformly suspend the yeast. The turbidity was determined in 1 cmcuvettes at 600 nm.

Inoculated tubes with no increase in turbidity were subcultured bytransferring 0.10 ml of the culture to 10 ml of fresh Medium 19 broth,incubating at 37° C. for 48 hours and recording the appearance of grossturbidity.

Growth Assays for Other Yeast and Bacteria.

The growth assay of S. cerevisiae and all bacteria except Lactobacilluswas assayed in a method parallel with C. albicans. All S. cerevisiaecultures were incubated at 25° C. Lactobacillus was cultured in MRSbroth instead of Medium 19.

The features and advantages of the present invention will be moreclearly understood by reference to the following examples, which are notto be construed as limiting the invention.

EXAMPLE I

Assay for Suppression by Glycerol, Glycols and Urea

A series of Medium 19 formulations was prepared containing eitherglycerol, ethylene glycol, propylene glycol, or urea as the supplement.The media were prepared from a stock of 2X Medium 19 broth and a 3 Msolution of the supplement. The supplement was diluted to a finalconcentration of 0 mM, 10 mM, 25 mM, 50 mM, 75 mM, 200 mM, 250 mM, 500mM, 750 mM, 1.00 M and 1.5 M. The control cultures contained aniso-osmotic concentration of NaCl. All preparations of media were steamsterilized. One set of each medium was prepared for each organism. Themedia were inoculated with C. albicans (ATCC 14053, wt), C. albicans(ATCC 38247), C. tropicalis, C. pseudotropicalis, C. stellatoidea, S.cerevisiae, Streptococcus sp (ATCC 19950), S. epidermidis, or E. coli.The growth of the organisms was assayed as described in the growth assayfor Candida.

A parallel assay was done for Lactobacillus sp (ATCC 9857) using MRSmedium as a nutrient medium in place of Medium 19 Broth.

Assay data is presented in Table II below for each of theyeast-destructive agents glycerol, ethylene glycol, propylene glycol,and urea, and for the sodium chloride control. For the various molarconcentrations of each, optical density values are shown at incubationtimes of 17, 46, 72, and 148 hours.

These data show that propylene glycol and urea were highly effective atconcentrations on the order of 1.5 M in suppressing the growth ofCandida tropicalis, while ethylene glycol and glycerol were lesseffective against this Candidal species. Similar data were obtained forcorresponding assays of Candida albicans.

These data also show that in some instances, e.g., in the case ofpropylene glycol and urea, increased incubation times and increasedconcentrations of the yeast-inhibitory agent coact to produce a highlyeffective removal of the undesired Candida species.

                                      TABLE II                                    __________________________________________________________________________    YEAST               ETHYLENE   PROPYLENE                                      DESTRUCTIVE                                                                            GLYCEROL   GLYCOL     GLYCOL    UREA       NaCL CONTROL              AGENT:   Concen-    Concen-    Concen-   Concen-    Concen-                   Incubation                                                                             tration,                                                                            Optical                                                                            tration,                                                                            Optical                                                                            tration,                                                                           Optical                                                                            tration,                                                                            Optical                                                                            tration,                                                                            Optical             Times, Hrs.                                                                            mM    Density                                                                            mM    Density                                                                            mM   Density                                                                            mM    Density                                                                            mM    Density             __________________________________________________________________________    17       0     1.213                                                                              0     1.149                                                                              0    1.262                                                                              0     1.13 0     1.115               "        100   1.103                                                                              100   1.073                                                                              100  1.147                                                                              100   1.151                                                                              125   1.057               "        1500  .911 1500  .631 1500 .013 1500  0    750   .822                 4       0     1.422                                                                              0     1.415                                                                              0    1.463                                                                              0     1.349                                                                              0     1.474               "        100   1.542                                                                              100   1.453                                                                              100  1.397                                                                              100   1.263                                                                              125   1.359               "        1500  1.33 1500  1.253                                                                              1500 0    1500  0    750   1.253               72       0     1.631                                                                              0     1.616                                                                              0    1.673                                                                              0     1.57 0     1.661               "        100   1.851                                                                              100   1.769                                                                              100  1.641                                                                              100   1.447                                                                              125   1.545               "        1500  1.632                                                                              1500  1.177                                                                              1500 0    1500  0    750   1.484               148      0     1.756                                                                              0     1.736                                                                              0    1.694                                                                              0     1.794                                                                              0     1.789               "        100   2.051                                                                              100   1.882                                                                              100  1.85 100   1.592                                                                              125   1.791               "        1500  1.836                                                                              1500  1.655                                                                              1500 0    1500  0    750   1.711               __________________________________________________________________________

                                      TABLE III                                   __________________________________________________________________________    YEAST               ETHYLENE   PROPYLENE                                      DESTRUCTIVE                                                                            GLYCEROL   GLYCOL     GLYCOL    UREA       NaCL CONTROL              AGENT:   Concen-    Concen-    Concen-   Concen-    Concen-                   Incubation                                                                             tration,                                                                            Optical                                                                            tration,                                                                            Optical                                                                            tration,                                                                           Optical                                                                            tration,                                                                            Optical                                                                            tration,                                                                            Optical             Times, Hrs.                                                                            mM    Density                                                                            mM    Density                                                                            mM   Density                                                                            mM    Density                                                                            mM    Density             __________________________________________________________________________    19       0     .193 0     .142 0    .144 0     .16  0     .14                 "        100   .111 100   .14  100  .094 100   .135 125   .131                "        1500  .029 1500  .031 1500 .019 1500  0    750   .007                24       0     .585 0     .564 0    .582 38    .142 0     .553                "        100   .524 100   .482 100  .558 100   .397 125   .45                 "        1500  .085 1500  .08  1500 .033 1500  0    750   .013                48       0     1.292                                                                              0     1.62 0    1.292                                                                              0     1.303                                                                              0     1.342               "        100   1.298                                                                              100   1.297                                                                              100  1.298                                                                              100   1.553                                                                              125   1.26                "        1500  1.075                                                                              1500  1.013                                                                              1500 549  1500  0    750   .058                72       0     1.224                                                                              0     1.554                                                                              0    1.238                                                                              0     1.243                                                                              0     1.235               "        100   1.2  100   1.203                                                                              100  1.218                                                                              100   1.486                                                                              125   1.227               "        1500  1.096                                                                              1500  1.158                                                                              1500 1.024                                                                              1500  0    750   .327                __________________________________________________________________________

The corresponding assay for S. cerevisiae, as representative of thenormal vaginal flora, is shown in Table III. This table is tabulatedanalogously with respect to Table II, except that the incubation periodsin Table III were 19, 24, 48, and 72 hours.

The data in Table III shows that the metabolic byproduct effect issignificant in the case of normal vaginal flora as well as the undesiredyeast species. To the extent that yeast species are present however, andare more affected by the yeast-inhibitory agent of the composition thanthe normal flora, the effects of byproduct-mediated reduction of thisnormal microbial population in the vagina will be significantly reduced.

Taken together, the data of Tables II and III show the suppressiveeffects of three byproducts compounds, glycerol, propylene glycol, andethylene glycol, as compared with the suppressive effects of equimolarconcentrations of urea and NaCl. The NaCl supplemented medium wasincluded as a control to evaluate osmotic effects. As shown in TableIII, NaCl did not affect the growth of either of the yeast organisms.Propylene glycol is a more potent inhibitor of yeast growth thanethylene glycol and glycerol was the least potent propylene glycol at1.5 M inhibited Candida. The other byproduct compounds were notinhibitory in the range assayed. Urea was an extremely potent inhibitorat >0.75 M.

In corresponding tests, the bacteria were insensitive to glycerol,propylene glycol and ethylene glycol at lower concentrations but weresensitive to high concentrations of each (FIG. 1). The bacteria wereless sensitive to the effects of urea than the yeasts. Lactobacillus sp(ATCC 9857) was the most sensitive of the bacteria tested. It wassuppressed by the highest level of glycerol and ethylene glycol.Propylene glycol at >1.5 M was inhibitory.

Glycerol suppresses growth of pathogenic, opportunistic and saprophyticyeasts at 1.5 M. It has little effect on the growth of bacteria normallyfound in the vaginal flora.

EXAMPLE II

Assay for Candidistatic and Candidacidal Concentration of Glycerol

The standard turbidimetric assay for C. albicans growth was used. TheMedium 19 broth based media were prepared from 10x Medium 19, glyceroland sterile water. Initially, the glycerol concentration varied from 0%to 75% in 23 increments. Turbidity was determined at 24, 28, and 48 hrs.At 48 hr., 0.20 ml was subcultured in regular Medium 19 broth. After 48hr. incubation, growth of C. albicans was completely inhibited by 15%glycerol. Subculture documented that glycerol concentrations of >30%were Candidacidal.

The experiment was repeated several times with glycerol concentrationranging from 0% to 30% in 2.5% increments and over a 96 hour reactioninterval. The data show that 15% glycerol caused a 50% suppression ofCandida growth for 96 hours and glycerol was Candidacidal at 25%. Thesaprophytic yeast, Saccharomyces cereviseae, was less susceptible tosuppression by glycerol and was not killed even at 30% glycerolconcentration.

EXAMPLE III

Effect of Intermediates on Vaginal Organisms

The effect of key metabolic products of intermediate energy metabolismon growth of vaginal organisms was surveyed. Compared with either aninert buffer control or a PO₄ buffer control, pathogenic, opportunisticand saprophytic yeasts were strongly inhibited by 0.10 M citrate atneutral pH and not effected by 0.10 M lactate or succinate between pH4.0 and pH 7.0. Growth of Staphylococci was inhibited by 75% by lactate,40% by acetate and completely by citrate at neutral pH. The E. coliculture was partially inhibited by lactate and acetate. There is thus amarked difference in the effect of key metabolic intermediates on theyeast and bacteria associated with vaginal flora. Candida was verysensitive to acetic acid concentration, while, in comparison bacteriahave little response at about pH 5 to pH 7.

EXAMPLE IV

Effect of pH on Growth of Vaginal Organisms

Respective samples of Medium 19 broth buffered with 200 mM2-N-Morpholino]ethanesulfonic acid (MES), Na phosphate, Na acetate, Nacitrate, and Na succinate were prepared. The appropriate amount ofacidic and basic forms of the buffers were calculated to provide pH 4.0,4.5, 5.0, 6.0, and 7.0 for 100 ml medium. 40 ml of water and 50 ml 2XMedium 19 broth were mixed and the pH determined. If necessary, the pHwas adjusted with 1 M NaOH or 1 M HCl and the final volume was adjustedto 200 ml. The control medium for each buffer was a mixture of 5 ml ofthe respective pH 6.0 medium, 45 ml water and 50 ml 2X Medium 19 broth,made to equal ionic strength with NaCl. All media were steam sterilized.

A rack of cultures consisting of all six buffered media at each of thefive pH values and a control was prepared to contain 9.9 ml/tube. Onerack of buffer tubes was prepared for each organism, C. albicans (ATCC14053), C. albicans (ATCC 38247), C. tropicalis, C. stellatoidea, S.cerevisiae, Streptomyces sp (ATCC 19950), S. epidermidis, and E. coli.Each tube was inoculated with 0.10 ml of the respective logarithmicphase culture diluted to A₆₀₀ =0.05. The tubes were mixed well andincubated. The growth of the organisms was determined turbidimetricallyat 600 nm at approximately 18, 24, 48, and 72 hours.

At the end of the incubation, the pH of the culture medium wasdetermined. If there was no increase in turbidity by the finaldetermination, 0.10 ml of the culture fluid was subcultured into regularMedium 19 broth, incubated and observed for growth after 48 hours.

A parallel experiment was done using the buffers diluted in MRS mediumand inoculated with Lactobacillus sp (ATCC 9857).

The results for Candida albicans, S. cerevisiae, and Lactobacillus areset forth below in Tables IV, V, and VI, respectively. The results foracetate are also shown graphically in the Figures as follows: forsampling at 23-30 hours post-infection for Streptococcus, E. coli andLactobacillus (FIG. 4); and for sampling at 24-32 and 46-48 hourspost-infection for C. tropicalis, C. stellatoides, C. albicans (strain14053) and S. cerevisiae (FIGS. 5-8 respectively). The results obtainedwith Candida albicans are generally representative of those obtainedwith the other Candida species, while the S. cerevisiae andLactobacillus are generally representative of normal vaginal floraspecies.

                                      TABLE IV                                    __________________________________________________________________________    CANDIDA                                                                       BUFFER:                                                                       MES          PO.sub.4 LACTATE                                                                              ACETATE  SUCCINATE                                                                             CITRATE                         Incubation                                                                            Optical                                                                              Optical  Optical                                                                              Optical  Optical Optical                       Times, Hrs.                                                                         pH                                                                              Denisty                                                                            pH                                                                              Denisty                                                                              pH                                                                              Denisty                                                                            pH                                                                              Denisty                                                                              pH                                                                              Denisty                                                                             pH                                                                              Denisty                       __________________________________________________________________________    18    4 .457 4 .427   4 .249 4 .018   4 .506  4 .811                          "     4.5                                                                             .553 4.5                                                                             .432   4.5                                                                             .46  4.5                                                                             3.000001E-03                                                                         4.5                                                                             .691  4.5                                                                             .662                          "     5 .599 5 .41    5 .581 5 .048   5 .661  5 .165                          "     6 .679 6 .36    6 .651 6 .275   6 .776  6 -.019                         "     7 .519 7 8.700001E-02                                                                         7 .72  7 .506   7 .683  7 .022                          "     6.1                                                                             .729 6.1                                                                             .677   6.1                                                                             .801 6.1                                                                             .7290001                                                                             6.1                                                                             .848  6.1                                                                             1.017                         24    4 1.058                                                                              4 .989   4 .876 4 .019   4 1.007 4 1.105                         "     4.5                                                                             1.141                                                                              4.5                                                                             .983   4.5                                                                             1.042                                                                              4.5                                                                             .011   4.5                                                                             1.195 4.5                                                                             1.058                         "     5 1.218                                                                              5 1.092  5 1.135                                                                              5 .058   5 1.145 5 .466                          "     6 1.151                                                                              6 .9690001                                                                             6 1.152                                                                              6 .75    6 1.232 6 .073                          "     7 1.104                                                                              7 .318   7 1.16 7 .899   7 1.114 7 .112                          "     6.1                                                                             1.219                                                                              6.1                                                                             1.196  6.1                                                                             1.264                                                                              6.1                                                                             1.134  6.1                                                                             1.213 6.1                                                                             1.202                         48    4 1.131                                                                              4 1.049  4 1.061                                                                              4 .017   4 1.147 4 1.171                         "     4.5                                                                             1.118                                                                              4.5                                                                             1.051  4.5                                                                             1.215                                                                              4.5                                                                             4.999999E-03                                                                         4.5                                                                             1.152 4.5                                                                             1.093                         "     5 1.107                                                                              5 1.098  5 1.183                                                                              5 .056   5 1.211 5 .8839999                      "     6 1.241                                                                              6 1.078  6 1.237                                                                              6 .809   6 1.13  6 .492                          "     7 1.085                                                                              7 1.1    7 1.199                                                                              7 .897   7 1.129 7 .614                          "     6.1                                                                             1.266                                                                              6.1                                                                             1.104  6.1                                                                             1.173                                                                              6.1                                                                             1.173  6.1                                                                             1.266 6.1                                                                             1.204                         72    4 1.294                                                                              4 1.164  4 1.052                                                                              4 .015   4 1.156 4 1.391                         "     4.5                                                                             1.315                                                                              4.5                                                                             1.062  4.5                                                                             1.198                                                                              4.5                                                                             .011   4.5                                                                             1.269 4.5                                                                             1.259                         "     5 1.246                                                                              5 1.305  5 1.267                                                                              5 .057   5 1.377 5 .8300001                      "     6 1.436                                                                              6 1.195  6 1.371                                                                              6 .786   6 1.324 6 .651                          "     7 1.202                                                                              7 1.292  7 1.261                                                                              7 .862   7 1.313 7 .657                          "     6.1                                                                             1.359                                                                              6.1                                                                             1.233  6.1                                                                             1.268                                                                              6.1                                                                             1.58   6.1                                                                             1.314 6.1                                                                             1.485                         __________________________________________________________________________

                                      TABLE V                                     __________________________________________________________________________    S. cerevisiae                                                                 BUFFER:                                                                       MES          PO.sub.4                                                                             LACTATE                                                                              ACETATE  SUCCINATE                                                                             CITRATE                           Incubation                                                                            Optical                                                                              Optical                                                                              Optical                                                                              Optical  Optical Optical                         Times, Hrs.                                                                         pH                                                                              Denisty                                                                            pH                                                                              Denisty                                                                            pH                                                                              Denisty                                                                            pH                                                                              Denisty                                                                              pH                                                                              Denisty                                                                             pH                                                                              Denisty                         __________________________________________________________________________    18    4 .038 4 .046 4 .035 4 .034   4 .041  4 .048                            "     4.5                                                                             .089 4.5                                                                             .08  4.5                                                                             .044 4.5                                                                             .005   4.5                                                                             .084  4.5                                                                             .032                            "     5 .063 5 .072 5 .092 5 .053   5 .061  5 .028                            "     6 .069 6 .066 6 .045 6 .045   6 .052  6 .024                            "     7 .057 7 .11  7 .044 7 .042   7 .071  7 .01                             "     6 .06  6 .064 6 .043 6 .07    6 .075  6 .057                            24    4 .396 4 .408 4 .391 4 .035   4 .373  4 .399                            "     4.5                                                                             .441 4.5                                                                             .467 4.5                                                                             .411 4.5                                                                             .018   4.5                                                                             .481  4.5                                                                             .345                            "     5 .488 5 .498 5 .56  5 .084   5 .471  5 .007                            "     6 .523 6 .526 6 .474 6 .438   6 .488  6 .01                             "     7 .398 7 .213 7 .457 7 .257   7 .384  7 .019                            "     6 .526 6 .515 6 .522 6 .536   6 .466  6 .456                            48    4 1.53 4 1.537                                                                              4 1.503                                                                              4 .021   4 1.493 4 1.512                           "     4.5                                                                             1.57 4.5                                                                             1.565                                                                              4.5                                                                             1.529                                                                              4.5                                                                             8.000001E-03                                                                         4.5                                                                             1.548 4.5                                                                             1.442                           "     5 1.556                                                                              5 1.54 5 1.556                                                                              5 1.052  5 1.506 5 .227                            "     6 1.591                                                                              6 1.554                                                                              6 1.529                                                                              6 1.409  6 1.528 6 .001                            "     7 1.536                                                                              7 1.314                                                                              7 1.551                                                                              7 1.33   7 1.468 7 .014                            "     6 1.616                                                                              6 1.576                                                                              6 1.579                                                                              6 1.57   6 1.585 6 1.529                           72    4 1.509                                                                              4 1.51 4 1.561                                                                              4 .025   4 1.529 4 1.519                           "     4.5                                                                             1.53 4.5                                                                             1.523                                                                              4.5                                                                             1.527                                                                              4.5                                                                             .299   4.5                                                                             1.497 4.5                                                                             1.452                           "     5 1.549                                                                              5 1.519                                                                              5 1.54 5 1.212  5 1.499 5 .359                            "     6 1.505                                                                              6 1.481                                                                              6 1.536                                                                              6 1.408  6 1.496 6 .053                            "     7 1.521                                                                              7 1.48 7 1.523                                                                              7 1.456  7 1.495 7 .021                            "     6 1.542                                                                              6 1.557                                                                              6 1.629                                                                              6 1.555  6 1.538 6 1.514                           __________________________________________________________________________

                                      TABLE VI                                    __________________________________________________________________________    LACTOBACILLUS                                                                 BUFFER:                                                                       MES          PO.sub.4 LACTATE  ACETATE  SUCCINATE                                                                             CITRATE                       Incubation                                                                            Optical                                                                              Optical  Optical  Optical  Optical Optical                     Times, Hrs.                                                                         pH                                                                              Denisty                                                                            pH                                                                              Denisty                                                                              pH                                                                              Denisty                                                                              pH                                                                              Denisty                                                                              pH                                                                              Denisty                                                                             pH                                                                              Denisty                     __________________________________________________________________________    18    4 0    4 0      4 0      4 0      4 0     4 0                           "     4.5                                                                             0    4.5                                                                             8.999999E-03                                                                         4.5                                                                             0      4.5                                                                             0      4.5                                                                             .083  4.5                                                                             .002                        "     5 .039 5 .069   5 .061   5 .029   5 .112  5 .046                        "     6 .142 6 .159   6 .23    6 9.000001E-02                                                                         6 .206  6 .153                        "     7 .331 7 .322   7 .79    7 .246   7 .371  7 .349                        24    4 0    4 0      4 0      4 0      4 0     4 0                           "     4.5                                                                             .004 4.5                                                                             .01    4.5                                                                             .005   4.5                                                                             0      4.5                                                                             .042  4.5                                                                             .006                        "     5 .054 5 .081   5 .078   5 .032   5 .109  5 .058                        "     6 .176 6 .172   6 .252   6 .106   6 .198  6 .172                        "     7 .984 7 .347   7 .796   7 .269   7 .376  7 .368                        48    4 0    4 0      4 0      4 0      4 .006  4 0                           "     4.5                                                                             .052 4.5                                                                             .021   4.5                                                                             .077   4.5                                                                             .002   4.5                                                                             .052  4.5                                                                             .023                        "     5 .733 5 .13    5 .61    5 .119   5 .177  5 .103                        "     6 1.2408                                                                             6 .842   6 .967   6 .798   6 .823  6 .2                          "     7 .984 7 .897   7 1.224  7 .921   7 .892  7 .414                        72    4 0    4 0      4 8.000001E-03                                                                         4 0      4 .004  4 8.999999E-03                "     4.5                                                                             1.393                                                                              4.5                                                                             .027   4.5                                                                             1.354  4.5                                                                             .051   4.5                                                                             .117  4.5                                                                             .082                        "     5 1.759                                                                              5 1.085  5 1.702  5 1.647  5 1.574 5 .111                        "     6 2.111                                                                              6 1.917  6 2.056  6 1.783  6 1.887 6 .245                        "     7 2.248                                                                              7 1.954  7 2.044  7 1.891  7 1.86  7 .456                        __________________________________________________________________________

Growth of the yeasts was generally not affected by pH in the range 4.5to 7.0. Representative bacterial flora associated with the vaginal tractwere strongly inhibited by acid pH and growth was nearly completelyinhibited below pH 6.0. A pH of 4.5 was inhibitory for most of thebacteria. The acetate buffer at pH 4.0 and 4.5 was inhibitory for allthree Candida species tested.

EXAMPLE V

Effect of Acetate/Acetic Acid Concentration on Growth of Vagina;Organisms

A stock of 2 M acetate buffer was prepared at pH 7.0 and 4.5. Two setsof tubes, one at pH 4.5 and one at pH 7.0, was prepared for eachorganism. One tube of each set-contained a final concentration ofacetate at 1 mM, 5 mM, 10 mM, 50 mM, 100 mM, 500 mM, and 1000 mM. A 4Xpreparation of Medium 19 broth was added to the proper volume of acetatebuffer and diluted to a final volume of 9.9 ml experimental medium at1X. The salts in the medium with 100 mM acetate were adjusted to 100 mMwith NaCl; then those with 100 mM acetate were adjusted to 1000 mM withNaCl.

One set of media was inoculated with each of C. albicans (wt), C.tropicalis, S. cerevisiae, S. epidermidis, and E. coli. The inoculum was0.10 ml of a logarithmic phase culture diluted to A₆₀₀ =0.05 with Medium19 broth. Each tube was mixed well and incubated. Growth was determinedturbidimetrically at 600 nm after 10, 16, 39, and 60 hrs. incubation.Cultures with no increase in turbidity at 600 nm were subcultured intoregular Medium 19 broth and observed for growth after incubation for 48hrs.

Data obtained during these tests for cultures of Candida albicans and S.cereviseae are set out in Tables VII and VIII below.

                  TABLE VII                                                       ______________________________________                                        CANDIDA                                                                              pH:                                                                           4.5           7.0                                                      Incubation                                                                             Concen-             Concen-                                          Times,   tration,  Optical   tration,                                                                              Optical                                  Hrs.     mM        Density   mM      Density                                  ______________________________________                                        10       1         .121      1       .184                                     "        5         .115      5       .19                                      "        10        .09       10      .178                                     "        50        .023      50      .175                                     "        100       .02       100     .159                                     "        500       .034      500     .085                                     "        1000      .05       1000    .122                                     16       1         .664      1       .836                                     "        5         .539      5       .827                                     "        10        .464      10      .789                                     "        50        .028      50      .748                                     "        100       .021      100     .737                                     "        500       .029      500     .111                                     "        1000      .044      1000    .124                                     39       1         1.302     1       1.393                                    "        5         1.377     5       1.406                                    "        10        1.217     10      1.406                                    "        50        .093      50      1.234                                    "        100       .024      100     1.225                                    "        500       .031      500     .222                                     "        1000      .053      1000    .135                                     60       1         1.591     1       1.787                                    "        5         1.63      5       1.817                                    "        10        1.5       10      1.645                                    "        50        .212      50      1.494                                    "        100       .042      100     1.228                                    "        500       .048      500     .373                                     "        1000      .071      1000    .158                                     ______________________________________                                    

                  TABLE VIII                                                      ______________________________________                                        S. CEREVISIAE                                                                        pH:                                                                           4.5           7.0                                                      Incubation                                                                             Concen-             Concen-                                          Times,   tration,  Optical   tration,                                                                              Optical                                  Hrs.     mM        Density   mM      Density                                  ______________________________________                                        10       1         .036      1       .051                                     "        5         .038      5       .052                                     "        10        .034      10      .053                                     "        50        .029      50      .061                                     "        100       .028      100     .065                                     "        500       .034      500     .087                                     "        1000      .05       1000    .12                                      16       1         .129      1       .121                                     "        5         .127      5       .134                                     "        10        .058      10      .129                                     "        50        .058      50      .135                                     "        100       .029      100     .129                                     "        500       .023      500     .077                                     "        1000      .044      1000    .113                                     39       1         1.541     1       1.542                                    "        5         1.518     5       1.545                                    "        10        1.445     10      1.523                                    "        50        1.166     50      1.542                                    "        100       .128      100     1.4                                      "        500       .042      500     .232                                     "        1000      .046      1000    .136                                     60       1         1.545     1       1.536                                    "        5         1.546     5       1.543                                    "        10        1.489     10      1.569                                    "        50        1.281     50      1.445                                    "        100       .254      100     1.445                                    "        500       .032      500     .093                                     "        1000      .061      1000    .129                                     ______________________________________                                    

Growth of the pathogenic C. albicans was 85% inhibited by 50 mM acetatebuffer at pH 4.5 and essentially completely inhibited at 100 mM acetatebuffer at pH 4.5. The acetate buffer at pH 7.0 had little effect. Thegrowth of the nonpathogenic yeast was suppressed by 100 mM acetatebuffer at pH 4.5. The acetate buffer at pH 7.0 had no effect on thegrowth of saprophytic yeast. The concentration of the acetate buffer perse had no effect on the growth of the representative vaginal bacteria.

EXAMPLE VI

Effect of Citrate Concentration on Growth of Vaginal Organisms

A stock of 4 M citrate buffer was prepared at pH 7.0 and 4.5. Two setsof tubes, one at pH 4.5 and one at pH 7.0, were prepared for eachorganism. One tube of each set contained a final concentration ofcitrate at 1 mM, 5 mM, 10 mM, 50 mM, 100 mM, 500 mM, and 1000 mM. A 4Xpreparation of Medium 19 broth was added to the proper volume of citratebuffer and diluted to a final volume of 9.9 ml experimental medium at1x. The salts in the medium with 100 mM were adjusted to 200 mM withNaCl; those with 100 mM citrate were adjusted to 2000 mM with NaCl.

The sets of medium at pH 4.5 and 7.0 were inoculated with each of C.albicans (wt), C. tropicalis, S. cerevisiae, S. epidermis, and E. coli.The inoculum was 0.10 ml of a logarithmic phase culture diluted to A₆₀₀=0.05 with Medium 19 broth. Each tube was mixed well and incubated.Growth was determined turbidimetrically at 600 nm after 10, 16, 39, and60 hours incubation. Cultures with no increase in turbidity at 600 nmwere subcultured into regular Medium 19 broth and observed for growthafter incubation for 48 hours.

The representative species of vaginal bacteria were much more sensitiveto citrate concentration than C. albicans. The saprophytic yeasts werenot effected by the citrate levels. Concentrations of citrate >500 mM ateither pH 7.0 or pH 4.5 were inhibitory for C. albicans.

The foregoing tests and data show the efficacy of the compositionsaccording to the present invention comprising an active yeast-inhibitoryagent in combination with a neutral buffering solution. The compositionsof the invention produce a fungistatic and/or fungicidal action on thevarious Candida species, and are highly effective in the treatment ofMonilia infections of the vagina. The neutral buffering of thecomposition of the invention permits vaginal flora to experience highgrowth rate, serving to effectively establish the normal biota of thevagina, following which its pH condition, as a result of metaboliteaccumulations, is gradually reduced to normal levels on the order ofabout 4.5, where increased concentration of the nonionized buffercompound further enhances the inhibitory activity of the composition. Tothe extent that the metabolic byproducts are effective in inhibiting theCandida species, the overall effectiveness of the composition is stillfurther enhanced.

EXAMPLE VII

Effect of Acetate Esters on Candida

Medium 19 tubes containing varying levels of methyl acetate, ethylacetate, diacetin or triacetin at about pH 7.0 were inoculated with C.albicans strains as described in Example VI, and turbidity wasdetermined periodically for 96 hours incubation. The results at 24hours, an example of which is shown in FIG. 2 as per cent suppression ascompared to controls, indicate an inhibitory effect of each ester ascompared to growth on Medium 19 alone.

EXAMPLE VIII

Effect of Serum on Glycerol Inhibition

Since transudation and exudation is characteristic of inflammatoryreactions and is frequently present in yeast infections, the impact ofserum on the ability of glycerol to inhibit yeast growth was determined.Serum concentrations from 0% to 10% were evaluated. Three types ofMedium 19 broth were prepared: regular; 10% glycerol added; and 20%glycerol added. Two sets of six culture tubes were prepared for eachtype of medium. To each medium, 0%, 0.5%, 1.0%, 2.5%, 5.0% or 10% calfserum was added to a tube of the series and the final volume was 9.9 ml.Each tube was inoculated with 0.10 ml logarithmic phase C. albicans orS. cerevisiae. The amount of growth was determined by turbidimetricassay at 600 nm at 16, 20, 40, and 48 hrs. FIG. 3 shows the results withCandida. A range of serum from 0 to 10% did not alter the inhibition ofCandida or Saccharomyces growth by 0 to 10% glycerol but increased theinhibition at 20% glycerol.

EXAMPLE IX

Effect of Nystatin on Yeast

Nystatin USP (Paddock Laboratories, Minneapolis, Minn.) was dissolved indimethyl formamide (DMF) at a concentration to 10 mg/ml DMF. Theputative activity was 5830 u/mg. A set of 10 assay tubes containing 9.9ml Medium 19 was prepared. A range of 1 to 100 units/ml finalconcentration of Nystatin was added to the medium in a total of 100 ulDMF. Controls containing 100 ul pure DMF and no DMF were also included.Each tube was mixed well and inoculated with 0.10 ml of logarithmicphase C. albicans or S. cerevisiae. The assays were incubated in thedark, C. albicans at 37° C., S. cerevisiae at 25° C. The turbidity ofthe cultures was determined at 24, 48, and 72 hours and at 7 days. Allassay tubes were subcultured at 96 hours by transferring 0.10 ml toregular medium 19 broth and incubating for 48 hours.

A concentration of 1 unit Nystatin/ml completely inhibited Saccharomycesat 72 hours and caused 50% suppression after 7 days incubation.Concentrations >1.5 units Nystatin/ml killed this saprophytic yeast.Concentrations >2.5 units Nystatin/ml were inhibitory to Candida.Candida was 99% inhibited by 2.5 units Nystatin/ml at 48 hours but only10% at 72 hours. Saprophytic yeast appear to be killed by about half theNystatin concentration required to kill C. albicans.

The effect of having 10% glycerol at the various Nystatin concentrationsin Medium 19 was to increase the inhibition of Candida. Thus, at any onesampling time, in the presence of glycerol, a lower concentration ofNystatin resulted in the same inhibitory effect as a higher level ofNystatin without glycerol.

Other anti-yeast antibiotics, such as clotrimazole, miconazole,butoconazole and terconazole may be used instead of Nystatin to augmentanti-yeast activity of the composition of the invention.

The following examples relate to the specific addition of Lactobacilluscells to the composition of the invention. In summary, in thisembodiment the invention comprises a composition having utility for thetreatment of vaginal yeast and as a vaginal cleanser while not adverselyaffecting normal vaginal bacterial growth and being capable of enhancinggrowth of vaginal bacteria as compared to growth of said bacteria at anormal vaginal pH, comprises the claimed acetic acid solution andLactobacillus species, and a method of allowing normal vaginal flora tobe restored, comprising douching with the composition. Preferably thecomposition comprises a solution comprising one or more medicallyacceptable agents, said agents selected from the group consisting ofethylene glycol, propylene glycol, glycerol, acetate derivatives ofglycerol, and acetate; said solution having active yeast-inhibitoryactivity and being effectively buffered with 10-500 mM acetate toestablish the pH of the composition from a pH above 4 to about pH 7; andcells of a Lactobacillus species. The Lactobacillus species ispreferably selected from the group consisting of Lactobacillusacidophilus, Lactobacillus jensenii, and Lactobacillus casei and mostpreferably is Lactobacillus acidophilus.

Preferably in the invention there is an effective concentration at thevaginal treatment site of at least 8% by volume of the compositionvolume plus the volume of other liquids present at the treatment site;and the acetate is selected from the group consisting of diacetin,triacetin, compounds of the formula CH₃ COOR where R is selected fromthe group consisting of hydrogen and C₁ -C₃ moieties, and salts ofacetic acid; and the active yeast-inhibitory agent and said effectivelybuffered aqueous acetate solution are in selected proportions relativeto one another effective to allow the active yeast-inhibitory agent tobe inhibitory against vaginal yeast of the genus Candida, and to enhancegrowth of vaginal bacteria as compared to growth of said bacteria at anormal vaginal pH.

Preferably the pH of the composition is at pH 5.01 to about pH 7.

EXAMPLE X

Effect of Adding Cells of Lactobacillus species in a Normal FloraInoculum

Normal flora including Lactobacillus were added to a simulated vaginalenvironment as discussed above which is equivalent to the extracellularfluid of the vaginal tissue, utilizing the composition of the inventionas described below.

In this experiment, the relative rate of growth and the finalcomposition of the vaginal flora, normal vaginal bacteria and pathogenicCandida albicans, were followed.

A flask containing about 50 ml standard M19 medium was inoculated with acolony of C. albicans ATCC#14053 and incubated at 37° C. for about 3hours. Small samples were aseptically withdrawn at 30 minute intervalsand the absorbency at 600 nm was determined, until the culture was instationary phase (no increase in absorbency for three successivemeasurements).

The normal flora inoculum was obtained from asymptomatic women asdiscussed herein and inoculated into two media, M19 broth and MRSLactobacillus broth, and the cultures were incubated at 37° C. for 18hours. A DeLong flask containing about 35 ml standard M19 medium wasinoculated with the overnight M19 culture, and a second flask containingabout 35 ml of MRS Lactobacillus broth was inoculated with the overnightculture grown in that medium. The optical density at 600 nm was plottedover time every 30 minutes. At stationary phase, equal volumes of thetwo cultures were mixed together to make the "normal flora inoculum" forthe experiment.

The two stationary phase inocula (Candida and normal flora) wereindividually diluted to an optical density of 0/050 at 600 nm usingEagle's MEM (2x)(Grand Island Biological Company). Two ml of eachinoculum was placed in a single sterile tube and mixed on a vortexmixer, and a 100 μl samples of the mixed inoculum was added to eachexperimental environment.

The experimental environments were (a) 100 mM MES, pH 7.00; (b) 100 mMacetate, pH 7.00; and (c) 100 mM acetate, pH 7.00 and 1 M propyleneglycol. The tubes of each environment were incubated at 37° C. as still,sealed aerobic cultures.

After 0, 6, 12, 18 and 24 hours of incubation, samples were asepticallywithdrawn. The number of viable organisms was determined by quantitativecounts on selective media. The samples were diluted 10-fold by sterilephosphate buffered saline to 10⁻⁸. The viable organisms were determinedby the standard pour plate method using three selective media, one eachfor Candida, Lactobacillus, and total aerobic bacteria.

Tubes of the basic plating agar were melted in the autoclave for aboutthree minutes. The medium was placed in a waterbath and was cooled andmaintained at 42° C. Each dilution was assayed in quadruplicate on eachselective medium. After addition of inoculum the tubes were poured intoplates, allowed to harden and incubated at 37° C. for 48 hours.

The results showed that the succession of Candida and normal vaginalflora in the two experimental environments utilizing acetate ((b) and(c)), as compared to the MES control (a), was statistically differentfrom the control at p<0.0001. The growth in the acetate/propylene glycolwas significantly different from the acetate treatment and from thecontrol treatment at a p value <0.0001. The analysis of variance for 160total cases of which 64 are missing (ln(x)):

    ______________________________________                                                    Sums of    Mean     F-                                            Source                                                                              df    Squares    Square   ratio   Prob                                  ______________________________________                                        Const 1     32152.2    32152.2  345278  ≦0.0001                        pH    2     23.4989    11.7495  126.18  ≦0.0001                        hr    3     312.818    104.273  1119.8  ≦0.0001                        ph * hr                                                                             6     3.19087    0.531811 5.7     ≦0.0001                        Error 84    7.82206    0.093120                                               Total 95    347.330                                                           Overall Mean: 18.30                                                           ______________________________________                                        Expected Cell Means of: 1n(x) on pH                                           Level of pH  Expected Cell Mean                                                                          Cell Count                                         ______________________________________                                        5            17.67         32                                                 6            18.35         32                                                 7            18.88         32                                                 ______________________________________                                        Coefficients of: 1n(x) on pH                                                  Level of pH                                                                             Coefficient                                                                              std. err. t Ratio                                                                              prob                                    ______________________________________                                        5         -0.6294    0.0440    -14.29 0.0000                                  6         0.0500     0.0440    1.135  0.2596                                  7         0.5794     0.0440    13.15  0.0000                                  ______________________________________                                    

EXAMPLE XI

Effect of Adding Cells of Lactobacillus acidophilus ATCC#9857

The invention may utilize the addition of vaginal Lactobacillusorganisms along with the composition. Lactobacillus cells used may bevaginal isolates or mixtures, or may be less recently isolated purecultures of Lactobacillus. Preferred species are L. acidophilus, L.jensenii, and L. casei, and the most preferred is L. acidophilus.

In the experiment reported herein, Lactobacillus acidophilus (ATCC 9857)and Candida grown to stationary phase as described above were each addedto tubes of the medium of the invention containing M19 Medium, 100 mMacetate alone, or 100 mM acetate and glycerol. The tubes were incubatedas described above for the experiment on normal flora. Tubes wereassayed for numbers of Lactobacillus and Candida as described.

Results are shown in FIG. 9. It is clear from this data that the levelsof Lactobacillus increase dramatically with all three treatments, whilethe numbers of Candida in the presence of the added Lactobacillusdecrease in both treatments as compared to the control.

EXAMPLE XII

Growth of Lactobacillus ATCC#9857 in MES, phosphate buffer, and acetate(100 mM)

Results of Lactobacillus ATCC#9857 growth and survival in MES ("Buffer1"), phosphate ("Buffer 2"), and acetate medium ("Buffer 3") accordingto the invention (100 mM acetate) with varying pH are shown in Table IX.Following the table is a presentation of the statistical analysis ofthese results showing that growth and survival of vaginal Lactobacillusat all pH values tested is significantly greater than at pH 4.0, withmost growth occurring in the acetate medium.

                  TABLE IX                                                        ______________________________________                                        Buf   pH    Hour   Lactobac.                                                                             Buf   pH  Hour  Lactobac.                          ______________________________________                                        1     4     7      9.3 × 10.sup.6                                                                  2     4   7     9.3 × 10.sup.6               1     4.5   7      9.3 × 10.sup.6                                                                  2     4.5 7     9.3 × 10.sup.6               1     5     7      1.0 × 10.sup.7                                                                  2     5   7     1.1 × 10.sup.7               1     6     7      1.3 × 10.sup.7                                                                  2     6   7     1.7 × 10.sup.7               1     7     7      2.2 × 10.sup.7                                                                  2     7   7     7.2 × 10.sup.7               1     4     10     9.3 × 10.sup.6                                                                  2     4   10    9.3 × 10.sup.6               1     4.5   10     9.4 × 10.sup.6                                                                  2     4.5 10    9.3 × 10.sup.6               1     5     10     1.1 × 10.sup.7                                                                  2     5   10    1.1 × 10.sup.7               1     6     10     1.5 × 10.sup.7                                                                  2     6   10    1.8 × 10.sup.7               1     7     10     2.4 × 10.sup.7                                                                  2     7   10    7.3 × 10.sup.7               1     4     23     9.3 × 10.sup.6                                                                  2     4   23    9.3 × 10.sup.6               1     4.5   23     1.1 × 10.sup.7                                                                  2     4.5 23    1.1 × 10.sup.7               1     5     23     6.2 × 10.sup.7                                                                  2     5   23    4.5 × 10.sup.7               1     6     23     2.1 × 10.sup.8                                                                  2     6   23    1.1 × 10.sup.8               1     7     23     1.2 × 10.sup.8                                                                  2     7   23    2.2 × 10.sup.8               1     4     48     9.3 × 10.sup.6                                                                  2     4   48    9.5 × 10.sup.6               1     4.5   48     3.4 × 10.sup.8                                                                  2     4.5 48    3.1 × 10.sup.8               1     5     48     8.7 × 10.sup.8                                                                  2     5   48    7.5 × 10.sup.8               1     6     48     2.2 × 10.sup.9                                                                  2     6   48    1.9 × 10.sup.9               1     7     48     3.1 × 10.sup.9                                                                  2     7   48    1.8 × 10.sup.9               3     4     7      9.3 × 10.sup.6                                       3     4.5   7      1.0 × 10.sup.7                                       3     5     7      1.2 × 10.sup.7                                       3     6     7      1.6 × 10.sup.7                                       3     7     7      2.4 × 10.sup.7                                       3     4     10     9.3 × 10.sup.6                                       3     4.5   10     1.1 × 10.sup.7                                       3     5     10     1.2 × 10.sup.7                                       3     6     10     1.6 × 10.sup.7                                       3     7     10     2.6 × 10.sup.7                                       3     4     23     9.4 × 10.sup.6                                       3     4.5   23     1.2 × 10.sup.7                                       3     5     23     1.5 × 10.sup.7                                       3     6     23     7.8 × 10.sup.7                                       3     7     23     9.3 × 10.sup.7                                       3     4     48     9.5 × 10.sup.6                                       3     4.5   48     1.3 × 10.sup.7                                       3     5     48     5.4 × 10.sup.8                                       3     6     48     1.2 × 10.sup.9                                       3     7     48     2.6 × 10.sup.9                                       ______________________________________                                    

The analysis of variance for 80 total cases (ln(x)):

    ______________________________________                                        Buffer 1 (MES):                                                                           Sums                                                              Source                                                                              df    of Squares Mean Square                                                                            F-ratio  Prob                                 ______________________________________                                        Const 1     24624.0    24624.0  2.4 × 10.sup.11                                                                  0.0001                               pH    4     70.1081    17.5270  1.7 × 10.sup.8                                                                   0.0001                               hr    3     171.656    57.2186  5.6 × 10.sup.8                                                                   0.0001                               ph * hr                                                                             12    53.1403    4.42836  4.3 × 10.sup.8                                                                   0.0001                               Error 60    0.000006   0.000000                                               Total 79    294.904                                                           ______________________________________                                        Expected Cell Means of: 1n(x) on pH                                           Level of pH  Expected Cell Mean                                                                          No. Obs.                                           ______________________________________                                        4            16.05         16                                                 4.5          16.98         16                                                 5            17.71         16                                                 6            18.39         16                                                 7            18.58         16                                                 ______________________________________                                        Sheffe's Post Hoc Test                                                        pH Comparison                                                                             Difference  Std. Error                                                                              Prob.                                       ______________________________________                                        4.5-4        0.934338   0.0001    0.000000                                    5-4         1.66683     0.0001    0.000000                                    6-4         2.34526     0.0001    0.000000                                    7-4         2.53419     0.0001    0.000000                                    All of the means are significantly larger as the pH is increased at           the 0.01 level.                                                               ______________________________________                                        Buffer 2 (Phosphate):                                                                     Sums                                                              Source                                                                              df    of Squares Mean Square                                                                             F-ratio Prob                                 ______________________________________                                        Const 1     24860.6    24860.6   3.1 × 10.sup.11                                                                 0.0001                               pH    4     92.3663    23.0916   2.84 × 10.sup.8                                                                 0.0001                               hr    3     130.485    43.4949   5.36 × 10.sup.8                                                                 0.0001                               ph * hr                                                                             12    38.5614    3.21345   3.97 × 10.sup.8                                                                 0.0001                               Error 60    0.000005   0.000000                                               Total 79    261.412                                                           ______________________________________                                        Expected Cell Means of: 1n(x) on pH                                           Level of pH  Expected Cell Mean                                                                          No. Obs.                                           ______________________________________                                        4            16.05         16                                                 4.5          16.97         16                                                 5            17.63         16                                                 6            18.31         16                                                 7            19.18         16                                                 ______________________________________                                        Sheffe's Post Hoc Test                                                        pH Comparison                                                                             Difference  Std. Error                                                                              Prob.                                       ______________________________________                                        4.5-4        0.920775   0.0001    0.000000                                    5-4         1.57459     0.0001    0.000000                                    6-4         2.25493     0.0001    0.000000                                    7-4         3.12483     0.0001    0.000000                                    All of the means are significantly larger as the pH is increased              at the 0.01 level.                                                            ______________________________________                                        Buffer 3 (100 mM acetate):                                                                Sums                                                              Source                                                                              df    of Squares Mean Square                                                                             F-ratio Prob                                 ______________________________________                                        Const 1     23773.1    23773.1   1.78 × 10.sup.11                                                                0.0001                               pH    4     75.5947    18.8987   1.4 × 10.sup.8                                                                  0.0001                               hr    3     90.1773    30.0591   2.25 × 10.sup.8                                                                 0.0001                               ph * hr                                                                             12    59.2596    4.93830   3.7 × 10.sup.8                                                                  0.0001                               Error 60    0.000008   0.000000                                               Total 79    225.032                                                           ______________________________________                                        Expected Cell Means of: 1n(x) on pH                                           Level of pH  Expected Cell Mean                                                                          No. obs.                                           ______________________________________                                        4            16.05         16                                                 4.5          16.24         16                                                 5            17.32         16                                                 6            18.06         16                                                 7            18.52         16                                                 ______________________________________                                        Sheffe's Post Hoc Test                                                        pH Comparison                                                                             Difference  Std. Error                                                                              Prob.                                       ______________________________________                                        4.5-4        0.183094   0.0001    0.000000                                    5-4         1.26508     0.0001    0.000000                                    6-4         2.00845     0.0001    0.000000                                    7-4         2.46256     0.0001    0.000000                                    All of the comparisons are significant.                                       ______________________________________                                    

While the invention has been described in detail with respect tospecific embodiments of the invention, it will be readily apparent thatother embodiments, modifications, and variations may be effectivelyemployed, and according, all such embodiments, modifications, andvariations are to be regarded as being within the spirit and scope ofthe invention.

What is claimed is:
 1. A method of allowing normal vaginal flora to berestored, comprising douching with a composition comprising a bufferedsolution of a yeast-inhibitory agent and cells of a Lactobacillusspecies, said yeast-inhibitory agent selected from one or more membersof the group consisting of ethylene glycol, propylene glycol, andglycerol, said solution buffered with 10-500 mM acetate to maintain thevaginal pH in the range of from pH above 4 to about pH 7, for sufficienttime to permit flushing of the vagina while permitting normal vaginalgrowth to occur in the vagina and enhancing growth of vaginal bacteriaas compared to growth of said bacteria a normal vaginal pH.
 2. Themethod of allowing normal vaginal flora to be restored, according toclaim 1, wherein said acetate is selected from the group consisting ofdiacetin, triacetin, compounds of the formula CH₃ COOR where R isselected from the group consisting of hydrogen, C₁ -C₃ alkyl moietiesand salts of acetic acid.
 3. The method of allowing normal vaginal florato be restored according to claim 1, wherein the active yeast-inhibitoryagent and said solution is formulated so that the activeyeast-inhibitory agent and the effectively buffered aqueous acetatesolution are in selected proportions relative to one another effectiveto allow the active yeast-inhibitory agent to be inhibitory againstvaginal yeast of the genus Candida, wherein growth of vaginal bacteriais enhanced as compared to growth of said bacteria at a normal vaginalpH.
 4. The method of claim 1, wherein the pH of the solution is at pH5.01 to about pH
 7. 5. The method of claim 6, wherein said Lactobacillusspecies is selected from the group consisting of Lactobacillusacidophilus, Lactobacillus jensenii, and Lactobacillus casei.
 6. Themethod of claim 1, wherein said Lactobacillus species is Lactobacillusacidophilus.
 7. A method of allowing normal vaginal flora to berestored, comprising applying to the vagina a composition comprising abuffered solution of a yeast-inhibitory agent and cells of aLactobacillus species, said yeast-inhibitory agent selected from one ormore members of the group consisting of ethylene glycol, propyleneglycol, and glycerol, said solution buffered with 10-500 mM acetate tomaintain the vaginal pH in the range of from pH above 4 to about pH 7,for sufficient time to permit flushing of the vagina while permittingnormal vaginal growth to occur in the vagina and enhancing growth ofvaginal bacteria as compared to growth of said bacteria a normal vaginalpH.
 8. A composition having utility for the treatment of vaginal yeastand as a vaginal cleanser while not adversely affecting normal vaginalbacterial growth and being capable of enhancing growth of vaginalbacteria as compared to growth of said bacteria at a normal vaginal pH,comprising:(a) a solution comprising one or more medically acceptableagents, said agents selected from the group consisting of ethyleneglycol, propylene glycol, glycerol, C₁ -C₂ alkyl acetate compounds,triacetin, diacetin, and acetate; said solution having activeyeast-inhibitory activity and being effectively buffered with 10-500 mMacetate to establish the pH of the composition from a pH above 4 toabout pH 7; and (b) cells of a Lactobacillus species.
 9. The compositionaccording to claim 8, wherein said Lactobacillus species is selectedfrom the group consisting of Lactobacillus acidophilus, Lactobacillusjensenii, and Lactobacillus casei.
 10. The composition according toclaim 8, wherein said Lactobacillus species is Lactobacillusacidophilus.
 11. The composition according to claim 8, wherein saidsolution comprises a medically acceptable active yeast-inhibitory agentselected from one or more members of the group consisting of ethyleneglycol, propylene glycol, and glycerol, and acetate;wherein there is aneffective concentration at the vaginal treatment site of at least 8% byvolume of the composition volume plus the volume of other liquidspresent at the treatment site; wherein said acetate is selected from thegroup consisting of diacetin, triacetin, compounds of the formula CH₃COOR where R is selected from the group consisting of hydrogen and C₁-C₃ moieties, and salts of acetic acid; and wherein the activeyeast-inhibitory agent and said effectively buffered aqueous acetatesolution are in selected proportions relative to one another effectiveto allow the active yeast-inhibitory agent to be inhibitory againstvaginal yeast of the genus Candida, wherein growth of vaginal bacteriais enhanced as compared to growth of said bacteria at a normal vaginalpH.
 12. The composition of claim 8, wherein the pH of the composition isat pH 5.01 to about pH 7.